Interpenetrating network design of bioactive hydrogel coatings with enhanced damage resistance

Bioactive hydrogel coatings offer a promising route to introduce sustained thromboresistance to cardiovascular devices without compromising bulk mechanical properties. Poly(ethylene glycol)-based hydrogels provide antifouling properties to limit acute thromobosis and incorporation of adhesive ligands can be used to promote endothelialization. However, conventional PEG-based hydrogels at stiffnesses that promote cell attachment can be brittle and prone to damage in a surgical setting, limiting their utility in clinical applications. In this work, we developed a durable hydrogel coating using interpenetrating networks of polyether urethane diacrylamide (PEUDAm) and poly(N-acryloyl glycinamide) (pNAGA). First, diffusion-mediated redox initiation of PEUDAm was used to coat electrospun polyurethane fiber meshes with coating thickness controlled by the immersion time. The second network of pNAGA was then introduced to enhance damage resistance of the hydrogel coating. The durability, thromboresistance, and bioactivity of the resulting multilayer grafts were then assessed. The IPN hydrogel coatings displayed resistance to surgically-associated damage mechanisms and retained the anti-fouling nature of PEG-based hydrogels as indicated by reduced protein adsorption and platelet attachment. Moreover, incorporation of functionalized collagen into the IPN hydrogel coating conferred bioactivity that supported endothelial cell adhesion. Overall, this conformable and durable hydrogel coating provides an improved approach for cardiovascular device fabrication with targeted biological activity.


Synthesis of PEGDA
Poly(ethylene glycol) diacrylate (PEGDA) was synthesized as described by Browning et al. with minor modifications. 7Briefly, triethylamine (2 equiv.) was added dropwise to a solution of PEG (3.4 kDa or 10 kDa, 0.1 mmol/mL; 20 kDa, 15wt%; 1 equiv.) in anhydrous dichloromethane under nitrogen atmosphere.Acryloyl chloride (4 equiv.) was added dropwise (1 drop every 4-5 s), and the reaction was stirred for 24 h.For fabrication of PEGDA 20 kDa, the reaction was allowed to stir for 48 h with additional acryloyl chloride (2 mole equivalents) added dropwise after 24 h.The reaction was then washed with potassium bicarbonate (8 equiv.)and dried with anhydrous sodium sulfate.PEGDA was precipitated in cold diethyl ether, filtered, and dried at room temperature overnight followed by vacuum drying.The degree of acrylation of the product was determined using proton nuclear magnetic resonance ( 1 H NMR) spectroscopy.Spectra were recorded on a Varian MR-400 400 MHz spectrometer and analyzed using a TMS/solvent signal as an internal reference.Polymers with percentage conversions of hydroxyl to acrylate end groups over 80% were used in this work. 1H-NMR (CDCl3): 3.6 ppm (m, -OCH2CH2-), 4.3 ppm (t, -CH2OCO-), 6.1 ppm (dd, -CH=CH2), 5.8 and 6.4 ppm (dd, -CH=CH2).

Table S1: First Network Effects on Mechanical Properties
Tensile properties PEUDAm single networks were determined at multiple molecular weights and concentrations relative to PEGDA networks.Increasing molecular weight resulted in significantly increased ultimate elongation, decreased ultimate tensile strength, and decreased modulus for PEGDA networks, Figure S2, (p < 0.0001, n = 12).For PEUDAm networks, increased molecular weight did not significantly affect ultimate tensile strength.Increasing macromer concentration for 20 kDa PEGDA networks resulted in significantly decreased ultimate elongation and increased modulus for PEGDA networks (p < 0.0001, n = 12); whereas, no statistical difference in ultimate tensile strength was observed.Increased macromer concentration for 20 kDa PEUDAm networks resulted in significantly decreased ultimate elongations, increased modulus, and increased ultimate tensile strengths (p < 0.005, n = 12).
Comparison of PEGDA and PEUDAm networks proved most differences to be insignificant, indicating high structural similarity between the hydrogels.A clear difference was the lower modulus of PEUDAm 3.4 kDa relative to PEGDA 3.4 kDa (97.2 ± 9.48 vs. 136 ± 11.1 kPa, p < 0001, n = 12, Figure S2).Ultimate elongations of these networks were not significantly different (38 ± 13 vs 40 ± 11%, n = 12), nor were swelling ratios (11 ± 0.21 vs. 11 ± 0.26, n = 12, Table S1).Additionally, PEUDAm 20 kDa had lower ultimate elongations than PEGDA 20 kDa at 10 and 15 wt% (170 ± 36 vs. 270 ± 52% at 10 wt%, p < 0.0001; 104 ± 30 vs. 190 ± 54% at 15 wt%, p < 0.001, n = 12, Figure S2).Secondary interactions between the urethane bonds near the acrylamide reactive groups are possible, though sterically hindered.However, if these interactions strongly affected network structure, it is likely clearer trends would have arisen in characterization.Due to the higher crosslinking density of the 3.4 kDa networks, it is possible that secondary interactions more strongly affect these hydrogels than higher molecular weights, resulting in modulus effects.Differences in 20 kDa networks could be caused by the dilute number of reactive groups in these systems leading to higher heterogeneity. 107Further characterization of network structure and likelihood of secondary interactions at crosslinking points would be necessary to determine any impact of the urethane motifs.

Figure S3 :
Figure S3: Tensile mechanical data comparisons for PEGDA and PEUDAm hydrogel networks as an effect of macromer molecular weight and concentration.A) Tensile modulus as an effect of n = 12 and error bars represent standard deviation.*** represents p < 0.001, and **** represents p < 0.0001.

Figure S7 :
Figure S7: Fracture properties of PEUDAm IPN networks.A) Representative curves.Effect of IPN on B) maximum force at break, C) elongation at break under fracture, and D) fracture energy.All comparisons represent an n = 12 and error bars represent standard deviation.* represents p < 0.05, and **** represents p < 0.0001.

Figure S8 :
Figure S8: FTIR characterization of bulk hydrogels and hydrogel coatings.A) pNAGA dry bulk hydrogel and NAGA monomer.B) IPN hydrogel coating compared to uncoated mesh and first network PEUDAm.